ABSTRACT: The overarching Goal of this proposal is to engineer a replacement airway for patients who must have their tracheas resected due to injury, infection, or cancer. Diseases of the trachea lead to approximately 4,000 tracheal excisions per year in the United States. But unlike most other connective tissues in the body - such as blood vessel, bone, skin and tendon - there currently are no replacements for tracheal tissue that are in widespread clinical use. For small tracheal defects that are less than ~ 5 cm in length, diseased tissue can generally be excised with primary re-anastomosis of the native trachea. However, this requires putting traction on the native airway that can lead to risk of ischemia, anastomotic dehiscence, and mediastinitis, which can be fatal. Furthermore, for longer tracheal defects, there is no approach at all to restore the airway. Therefore, lack of a suitable tracheal replacement is a Significant medical problem. The ideal replacement tracheal tissue would be one that has the mechanical properties of the native airway (eg. can resist both tensile and compressive forces); does not require immunosuppression; is easily implantable using standard techniques; can survive the tenuous blood supply of the tracheal environment without anastomotic failure; and is readily available. Recently, we described a novel engineered, acellular tissue that fulfills most of these requirements, and which functions for at least two months in several animal models. However, in approximately 30% of implants in rodents and primates, we have observed mid-graft fibrotic stenoses that led to airway occlusion, which contained fibroblasts and abundant collagen matrix deposition, but little epithelial repopulation. The underlying Premise of this application is that undesirable host remodeling responses lead to the airway fibrosis and stenosis that is seen in a subset of these engineered tracheas. Specifically, we hypothesize that a trigger of host fibrosis may be the supra-physiological stiffness of the engineered airways, which can impact the Hippo pathway and TGF-?signaling and lead to fibroblast proliferation and collagen deposition. A second hypothesis is that inadequate epithelial re-population of the engineered trachea by host basal cells may lead to a lack of local inhibitors of fibrosis, including prostaglandin E2. This application will explore both of these hypotheses in efforts to improve the long-term functionality of engineered tracheal replacements. In the long term, the Impact of this work relates to developing a functional tracheal replacement that could help thousands of patients each year. In addition, we may Impact our understanding of tracheal stenosis that occurs in other clinical settings.